From our investigation, the NCF had also identified several additional patents that
yielded new information that hadn't been seen by other scientists or physicians in the
field. In one patent [23], Dr. DeMeirleir and his colleagues expanded the RNase L
fragmentation model in CFS and provided descriptions for their scientific discoveries: "The
lower molecular weight fragments of RNase L are produced from the specific cleavage of
native RNase L by a protease(s). These fragments then play a selective role in inhibiting
the apoptotic pathway, in effect keeping the damaged cell alive and the immune system
dysfunctional." The researchers had identified three RNase L fragments.
In Fragment #1, they found an ankyrin binding repeat domain which is known to interact
with various transport proteins. In addition, they also identified sequence homology with NF-kappaB (Nuclear transcription Factor kappa B).

NF-kappaB has been demonstrated to induce transcriptional activities within a cell to
promote cell growth thus acting in an anti-apoptotic manner. In Fragment #2, they
identified the 2-5A binding fragment that has catalytic activity and thus is able to
degrade RNA. Since this fragment competes with the native RNase L protein for free 2-5A,
it may be responsible for inhibiting the complete induction of apoptosis. They found that
Fragment #3 shared homology with chain A of Cdk6 (Cyclin dependent kinase). Cdk6 acts to
block apoptosis by altering the cell cycle. In fact, the inventors provide a diagram
(Figure 1) that explicitly shows the role of these RNase L fragments in CFS pathogenesis.
Since the 2-5A synthetase / RNase L pathway is part of the antiviral defense mechanism in
cells and because it plays a role in the regulation of cell growth / differentiation,
these RNase L fragments provide critically important insight into the cellular mechanisms
that are uniquely responsible for the immune dysfunction in CFS patients.

Furthermore, the inventors conclude that "The presence (or absence) of low
molecular weight RNase L fragments 1 to 3, and optionally the caspase activity data, is
then used to diagnose whether or not the host suffers from the chronic immune
disease." Caspases are cysteine-aspartic acid proteases that are considered to
be the cell death executioners due to the absolute requirements for their presence in cell
death / apoptosis [24].

Extensive analysis was done by Dr. DeMeirleir and his colleagues in this patent. They
studied four patients who had been administered ampligen for a period of at least six
months. These patients were also compared with healthy controls and forty-four additional
patients who had not undergone ampligen therapy. All patients were evaluated for RNase L
fragmentation and caspase levels. For the 44 patients, when the RNase L ratios were 6 to 8
(6 < RNase L ratio < 8), the caspases C2, C3, C6, C8, and C9 reached their peak.
Caspase levels were generally lower overall for lower RNase L levels (RNase L ratio <
6) as well as for higher RNase L levels (RNase L ratio > 8). In fact the inventors
concluded "Upon further analysis, it is evident that levels of all caspases
assayed (C3, C3, C6, C8, and C9) at first increase, then decrease relative to the RNase L
ratio in CFS patient groups P1 through P7, indicating that the apoptotic process is being
inhibited when the levels of RNase L-related fragments reaches a certain point."
In their evaluation of four patients treated with ampligen, two patients responded to
therapy in that their RNase L ratio decreased and they experienced increases in their
Karnofsky performance scores (KPS). The Karnofsky performance test measures an
individual's ability to function and carry on normal activities. Karnofsky scores range
from zero for a nonfunctional or dead patient to 100 for a completely normal person. The
third patient did not respond to ampligen due to an increase in the RNase L ratio and a
decrease in Karnofsky score. The last patient responded to ampligen initially with
reductions in RNase L ratio and an increase in Karnofsky score. However, six months after
cessation of therapy, the patient suffered a relapse as indicated by a decrease in
Karnofsky score and a significant increase in the RNase L ratio. Interestingly, for this
patient, the RNase L ratio after the cessation of therapy and subsequent relapse was much
greater (52.9) than even before beginning therapy (8.7). In both the non-responder and
relapsed patient, low levels of caspase-3 (C3) that were found may indicate that a block
exists in the apoptotic pathway.

Dr. DeMeirleir and colleagues, in this patent, concluded that (A) Increases or
decreases in the relative amounts (i.e., ratios) of native RNase L when measured against
RNase L-related fragments correlates strongly with the presence or absence of CFS disease,
respectively; (B) Increases in apoptosis in PBMCs from CFS patients can be measured by
analyzing caspase levels; (C) Increases in apoptosis in PBMCs from CFS patients can be
measured by analyzing the relative amount of native RNase L and related fragments; (D) As
the ratio of RNase L-related fragments to the remaining native RNase L protein increases
above a certain level, the process of apoptosis, as measured by caspase levels, appears to
stop then decline even further to sub-normal levels; (E) RNase L-related fragments are
likely to inhibit the apoptotic process based on the amino acid sequence comparison of
RNase L-related fragments to known inducers of cell activation and growth; (F) Upon
successful therapy with mismatched double stranded RNA (i.e. ampligen), RNase L ratios
return to 'normal' or healthy control levels; (G) Upon successful therapy with mismatched
double stranded RNA, caspase levels return to 'normal' or healthy control levels; (H)
Mismatched double-stranded RNA induces the synthesis of 2'-5'A. In turn, 2'-5'A binds to
and activates native RNase L homodimers that in turn induce apoptosis, removing the
anti-apoptotic block, allowing for return of normal cellular functions.

However, Dr. DeMeirleir and colleagues have provided exciting information regarding a
potential treatment therapy identified in their newest patent. The patent, titled
"Methods of treatment of chronic immune diseases," was just issued on July 31,
2003 [25]. In this patent, the inventors detailed their findings regarding
treatment for multiple sclerosis and chronic fatigue syndrome, both of which the inventors
consider to be chronic immune diseases. Treatment includes administering to a human an
effective amount of a protease inhibitor where this protease inhibitor is an elastase
inhibitor and where the elastase inhibitor is a beta-lactam containing compound such as
the cephem cefoperazone. Furthermore, the inventors state that the diagnosis
of chronic immune disease is accomplished by detecting the presence of RNase L fragments.
This comes as no surprise because of the volumes of previous work on RNase L as well as
the inventors previous patents. In fact, another research group has recently published
that RNase L ratios could distinguish CFS patients from healthy controls [26].

Briefly stated, Dr. DeMeirleir and colleagues demonstrated that the enzyme elastase is
able to generate fragments of recombinant RNase L protein, the size of which approximates
the fragment of native RNase L protein found in peripheral blood mononuclear cells from
patients with MS and CFS. This is in-line with a previous publication by DeMeirleir and
others [27] where RNase L proteolysis could be mimicked by combining recombinant RNase L
with human leukocyte elastase. In the patent, the inventors were able to demonstrate that
pure / recombinant RNase L (> 95%) was proteolytically cleaved by elastase and the
fragments of RNase L generated by elastase digestion were compared to the size of the
fragments of native RNase L found in PBMCs from CFS patients. The inventors demonstrated
that the pure RNase L cleavage product (37 kDa) was equivalent in size to that found in
CFS patients. The inventors then tested the effects of the beta-lactam based
antibiotic cefoperazone (Pfizer Pharmaceuticals, trade name Cefobid) on the levels of
RNase L protein in the human monocytic leukemia cell line U937. The inventors demonstrate
that cefoperazone is able to inhibit the production of the low-molecular weight fragment
of native RNase L protein and that this inhibition is dose dependent. They also
demonstrate that cefoperazone is able to inhibit elastase activity as indicated by a
reduction of low-molecular weight RNase L protein by in-vitro testing. This
indicates that beta-lactam based antibiotics have an effect on elastase, via its
inhibition, and thereby are able to reduce fragmentation associated with RNase L cleavage.
In fact, from the previous publication, the authors state that enhanced human
leukocyte elastase activity appears to be involved in the increased proteolysis of RNase L
in CFS PBMCs. Since the inventors listed numerous antibiotics that contain
beta-lactam compounds, without discussing their targeted sensitivity, this could help
explain why some patients are at least partially responsive to certain antibiotics and why
they have reported that they feel better on antibiotic therapy. If their antibiotics
contain beta-lactam based compounds then elastase would be inhibited and this would
potentially move the patient in the direction of native RNase L restoration by reducting
the cleavage products!

A brief look by the NCF into the role(s) of human leukocyte elastase provided us with
answers to many additional questions that we had. One of these was the fact that elastase
has been found in significant amounts in several different types of amyloidoses [28] and
in amyloid fibrils themselves [29,30]. The NCF had noted previous research that examined
the molecular basis of CFS [31]. In this work, a unique urinary marker contained
N-methylpyrrolidine and was found to be highly significant in CFS patients. However,
N-methylpyrrolidine has already been found to accelerate the aggregation process
associated with amyloid formation [32]. These findings strengthen our NCF hypothesis for
potential amyloid involvement and protein misfolding in the pathogenesis of CFS.

Elastase has been found to cleave all six isulin-like growth factor binding proteins
(IGFBPs) in-vitro and in-vivo with a significant proteolytic cleavage of IGFBP-3 [33].
IGFBP's are associated with the proliferative effects of insulin-like growth factors
(IGFs) on various cells. Interestingly, IGFBP-3 has been found to upregulate STAT1 and to
increase its protein

expression [34]. Acclydine therapy, as suggested by Dr. DeMeirleir, acts by increasing
IGFBP-3 [19]. Thus, by increasing the levels of IGFBP-3 in the cell, IGF-1 is blocked from
binding to its receptor thereby suppressing the growth of the cell, promoting apoptosis,
and counteracting the loss of functional p53 protein on the growth of the cell.
Additionally, lycopene, which is a natural carotenoid found in tomatoes and readily
available as a supplement, has also been found to increase IGFBP-3 as well [35,36].

Elastase has been implicated in chronic inflammation [37] including rheumatoid
arthritis [38] and is the target of antirheumatic drugs [39]. In fact, structural changes
have been observed in the skin [40] by elastase and in the kidney [41] elastase mediates
glomerular injury in-vivo causing proteinuria due to changes in glomerular permeability.
Elastase has also been found to cleave the T4-binding globulin (TBG) [42]. TBG serves to
maintain an important serum pool of thyroid hormones and to prevent their excessive loss
in urine. Elastase also cleaves the corticosteroid-binding globulin reducing its
hormone-binding affinity and capacity [42]. In addition, elastase is involved in
fibrinolysis [43] as it has been found to degrade fibrin and inhibit the blood coagulation
system by degrading key proteins.

However, other interesting findings for elastase are that it regulates Stromal
cell-derived factor-1 (SDF-1)/CXCR4 binding [44]. This is particularly intriguing because
Human Herpes Virus-6 (HHV-6) uses the CXCR4 receptor for infection [45]. HHV-6 has been
implicated in the pathology for both multiple sclerosis and chronic fatigue syndrome [46].
Furthermore, elastase has been found to cleave CD4 (helper cells) and CD8
(cytotoxic/suppressor cells) lymphocytes leading to a reduction in number of these cell
types [47]. In addition, elastase has also been found to cleave immune complexes and to
regulate inflammation by a feedback mechanism that can lead to cyclic inflammatory states
[48].

The finding that Dr. DeMeirleir and colleagues found beta-lactam antibiotics to be
associated with the inhibition of human leukocyte elastase is echoed by various
publications [49]. However, the NCF has identified one sensitive inhibitor of leukocyte
elastase that is readily available and it is boswellic acid [50].

Dr. DeMeirleir's colleagues also have a recent patent application for the cleavage of
actin [51]. In this application, the inventors found correlations between the relative
amount of native RNase L protein in PBMC extracts and the relative amount of native actin
protein in serum. They also found correlations between the ratio of RNase L fragments and
the ratio of actin fragments. Actin is a protein that assists in the diverse activities of
the cytoskeleton of the cell which includes cell signalling. Interestingly, elastase has
been shown to cleave actin in Behcet's disease, a disorder that involves inflammation of
the blood vessels [52].

In conclusion, it is obvious that Dr. DeMeirleir and his colleagues, in cooperation
with other medical researchers, have investigated several key mechanisms in the
pathogenesis of CFS and MS and these included STAT1, RNase L cleavage, actin, and the use
of elastase inhibitors as a potential therapy for these chronic diseases. Hopefully by
highlighting this research and explaining how it fits within a larger disease framework,
the NCF has acted in a responsible

manner to inform, educate, and to provide much needed hope to patients, their
physicians, and to researchers worldwide.

Use of leukocyte elastase inhibitors may potentially treat patients with CFS as well as
MS due to its impact on RNase L and STAT1.

Beta-lactam based antibiotics act as leukocyte elastase inhibitors, however drug
sensitivity is most likely unknown for this target.

The National CFIDS Foundation, Inc. provides informative and up-to-date quality
scientific research assessments for the CFS patient community, treating physicians, and
medical researchers alike. By utilizing evidence-based medical techniques which
incorporate the judicious use of intellectual property rights in conjunction with
traditional disease profiling methods, investigational reports are generated with the
explicit intent of validating the clinical importance and applicability of a discovery as
well as to assist in propelling appropriate CFS medical research forward.

Additional details on all of our research activity and scientific results will be
discussed in future issues of The Forum and updated on our website at www.ncf-net.org. We
greatly appreciate all donors and supporters of the NCF's Research Grant Program. Without
your financial support, we would be unable to appropriately fund the aggressive research
mentioned in this article. However, since this research is ongoing, please consider
continued donations to the NCF's Research Grant Program to support these efforts. The
National CFIDS Foundation, Inc. is the largest, all volunteer, and fastest growing
non-profit CFIDS organization in the U.S. Because we have no paid staff, 100% of all
donations go directly to research. Please continue to help us help you by supporting us
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